Abstract
Human dental pulp stem cells (hDPSCs) are the primary cells responsible for dentin regeneration. Typically, in order to allow for odontoblastic differentiation, hDPSCs are cultured over weeks with differentiation-inducing factors in a typical monolayered culture. However, monolayered cultures have significant drawbacks including inconsistent differentiation efficiency, require a higher BMP concentration than should be necessary, and require periodic treatment with BMPs for weeks to see results. To solve these problems, we developed a 3D-cell spheroid culture system for odontoblastic differentiation using microparticles with leaf-stacked structure (LSS), which allow for the sustained release of BMPs and adequate supply of oxygen in cell spheroids. BMPs were continuously released and maintained an effective concentration over 37 days. hDPSCs in the spheroid maintained their viability for 5 weeks, and the odontoblastic differentiation efficiency was increased significantly compared to monolayered cells. Finally, dentin-related features were detected in the spheroids containing BMPs-loaded microparticles after 5 weeks, suggesting that these hDPSC-LSS spheroids might be useful for dentin tissue regeneration.
Highlights
In a normal biological system, stem cells undergo intensive cell-cell contacts to develop tissues with three-dimensional (3D) structures
Spheroid culturing of human periodontal ligament stem cells significantly enhanced stemness and osteogenic potential compared with hPDLSCs cultured in a monolayer [11, 20]
We demonstrated that the microparticles with the leaf-stacked structure (LSS) significantly prevent cell necrosis in human bone marrow-derived mesenchymal stem cells
Summary
In a normal biological system, stem cells undergo intensive cell-cell contacts to develop tissues with three-dimensional (3D) structures. This is mediated by the microenvironment including the extracellular matrix surrounding cells, which are distinct physiological cues for cell differentiation and proliferation. The normal physiological behaviors of stem cells can be distorted in monolayer cultures, leading to the loss of the multilineage potential and replicative ability [2, 3]. To mimic the physiological environment, various methods have been reported for culturing cells in a 3D structure, including scaffold-free cultures [4, 5], cultures grown on various scaffold materials [6,7,8], and cultures embedded in gel materials [9, 10]. Osteo/odontoblastic gene expression and mineralization are upregulated in immortalized mouse
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